Fluorescence allows for the detection of very low amounts of fluorescent molecules due to a very high signal-to-noise ratio (the background is typically non-fluorescent). Fluorescent colloids—from nano to microns size particles are used in a broad range of applications involving tagging, tracing, labeling (Hasegawa et al., 2005, Edwards et al., 2004, Lizard et al., 2004, Meldal, 2002, Ohata et al., 2003, Iyer et al., 2009, Tan et al., 2004), and particularly in biological applications (Halas, 2009). Using various reactive moieties such as carboxylic acids, biotin, streptavidin, amine, thiol, maleimide, succinimide, etc., one can attach specific sensing molecules to a labeling colloid, such as antibodies, various proteins, peptides, nucleic acids, aptamers, small molecules, and even liposomes. The particles could be bioconjugated with multiple biomolecules for multimodal, multiplexed imaging of large molecules, cells, tissues, and animals (Chen et al., 2009, Doering and Nie, 2003, Dupuy et al., 2005, Eastman et al., 2006, Fritzler, 2006, Gao and Dave, 2007, Gao et al., 2005, Gonzalez-Buitrago and Gonzalez, 2006, Jokerst et al., 2009, Lauer and Nolan, 2002, Liew et al., 2007, Mirasoli et al., 2009, Wang and Tan, 2006a, Wang et al., 2005, Wang et al., 2007).
General Approach to Synthesize Fluorescent Colloidal Particles.
Fluorescence of colloidal particles is typically achieved through incorporating either inorganic or organic fluorescent dyes (or pigments, quantum dots) into the particle's material. While inorganic dyes are typically more stable, their limited variety, relatively low quantum yield, and compatibility are the issues restricting their broad application. Large varieties of organic dyes and their high quantum yield, make them attractive to be used in fluorescent particles. However, the problems of organic dyes are in their low photostability and frequent change of their fluorescence dependence on the chemistry of the environment. Incorporation of dyes into silica matrix seems to be one of most promising approaches because of the excellent sealing ability and wide compatibility of silica with other materials, including biocompatibility. Numerous attempts to embed organic dyes into silica xerogels and zeolites have reported (Rao and Rao, 2003, Klonkowski et al., 2002, Deshpande and Kumar, 2002, Leventis et al., 1999, del Monte and Levy, 1998, Suratwala et al., 1998, Calzaferri et al., 2003, Zhao et al., 2004, Santra et al., 2004). Silica has been material of choice due to its good biocompatibility, low toxicity, and ease of functionalization with sensing molecules.
To prevent leakage of the dyes out of the porous matrix of xerogels, dyes were typically covalently bound to the silica matrix (Audebert et al., 1996, Bagwe et al., 2004, Frantz et al., 2002, Leventis et al., 1999, Baker et al., 1999, Suratwala et al., 1998, Lin et al., 2005, Antonini et al., 2000). While the photostability of such materials is higher than the stability of pure dyes, it typically does not prevent bleaching substances (including oxygen), from penetration inside such a composite material. Moreover, in the case of xerogel, it is rather hard to make well defined particles out of xerogel, which prevents it from being used as labels. Fluorescent lasing dyes possess relatively high photostability and excellent quantum yield. Incorporation of such dyes into mesoporous patterned silica films (Yang et al., 2000) and silica rods (Marlow et al., 1999) to create a new laser material has been reported.
Fluorescent particles are widely manufactured, but the processes used for their production are often tightly held trade secrets. So far the brightest particles have been made of quantum dots incorporated into polymer matrix (See Han, et al. 2001). Incorporation of dyes and quantum dots into glass particles seems to be one of most promising because of excellent sealing ability of the glass and wide compatibility of glass with other materials.
High brightness of labeling silica particles is desirable to attain higher signal-to-noise ratio, and consequently, to increase the sensitivity and/or speed of detection. There have been many attempts to make fluorescent nanoparticles silica of high brightness (Shibata et al., 1997, Ow et al., 2005, Larson et al., 2003, Bagwe et al., 2004, Wang and Tan, 2006b, Zhao et al., 2004, Yang et al., 2003, Kim et al., 2006). Most of the approaches utilized the covalent coupling between fluorescent dye and silica. Recently, the use of tris(2,2A-bipyridyl)dichlororuthenium(II) hexahydrate (Rubpy) dye was reported to non-covalently dope silica nanoparticles (Santra et al., 2001). All of these particles showed brightness comparable with a single bright quantum dot at best.
Another approach, the synthesis of one-step self-assembly of nanoporous (sometimes called mesoporous) silica particles with physically encapsulated organic dyes was proposed (Naik and Sokolov, 2008, Sokolov et al., 2007) by the applicant. It is a templated sol-gel self-assembly of nanoporous particles with fluorescent dye added in a relatively large concentration (up to 0.01M) to the synthesizing bath. After completing the synthesis, the dye stays inside of self-sealed cylindrical nanochannels. The particles were up to two orders of magnitude brighter than polymeric particles of the same size assembled with quantum dots (ZnS-capped CdSe quantum dots) (Santra et al., 2001) (we called these particles “ultrabright” for the lack of a better term).
However, the particles synthesized in (Naik and Sokolov, 2008, Sokolov et al., 2007) were colloids of several microns in size, whereas most of bio-labeling application require nanosize particles. The efforts to scale the particles down to nanoscale were done in the by Sokolov and Naik (Sokolov and Naik, 2008). The reported brightness of ˜30 nm silica mesoporous particles was so far only ˜40% of the brightness of a capped water dispersible quantum dot (about the size of 5-60 nm (Biju et al., 2010) dependent on the fluorescent wavelength. In the same time, a simple geometrical extrapolation of the brightness attained in the micron size particles to the brightness of 30 nm silica particles gives us the value which is substantially higher than the brightness of a quantum dot. The culprit was presumably in the dye leaking out of the nanoparticles.